Split‐Ubiquitin System for Identifying Protein‐Protein Interactions in Membrane and Full‐Length Proteins

Christopher Grefen1, Sylvie Lalonde2, Petr Obrdlik3

1 Universität Tübingen, Tübingen, Germany, 2 Carnegie Institution, Stanford, California, 3 IonGate Biosciences GmbH, Frankfurt, Germany
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.27
DOI:  10.1002/0471142301.ns0527s41
Online Posting Date:  October, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Protein‐protein interactions play a fundamental role in the regulation of almost all cellular processes. Thus, the identification of interacting proteins can help to elucidate their function. The mating‐based split‐ubiquitin system (mbSUS) uses yeast as a test organism to identify potential interactions between full‐length membrane proteins or between a full‐length membrane protein and a soluble protein. The mbSUS can also be used to provide further evidence for protein‐protein interactions detected with other methods and to map the interaction domains of selected proteins. The mbSUS is optimized for systematic screening approaches employing a mating‐based approach, as typically used to determine protein interactions on a genomic scale. Construction of bait and prey fusions is simplified by adapting two different cloning procedures: (i) in vivo cloning in yeast, and (ii) Gateway cloning in E. coli. Protocols for small‐scale interaction tests, as well as systematic approaches using sorted bait and prey arrays, are described. Curr. Protoc. Neurosci. 41:5.27.1‐5.27.41. © 2007 by John Wiley & Sons, Inc.

Keywords: Split‐ubiquitin; mbSUS; protein‐protein interaction; full‐length membrane protein; mating; systematic analysis; Gateway

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Cloning of Cub‐PLV and Nub Fusion Constructs
  • Basic Protocol 2: Characterization of Mating‐Based Split‐Ubiquitin System Bait Fusions
  • Basic Protocol 3: Systematic Split‐Ubiquitin Interaction Tests with Selected Bait and Prey
  • Alternate Protocol 1: Cloning of Mating‐Based Split‐Ubiquitin Systems Fusions in E. coli
  • Alternate Protocol 2: Systematic Screening of Sorted Collections of Split‐Ubiquitin Bait and Prey Fusions
  • Alternate Protocol 3: Quantitative Analysis of Interactions Between Cub‐PLV and Nub Fusions
  • Support Protocol 1: Use of Different Mating‐Based Split‐Ubiquitin System Nub Vectors for Interaction Tests
  • Support Protocol 2: Plasmid Rescue from Yeast: “Lazy Bones” Protocol
  • Support Protocol 3: Preparation of Protein Extracts for Immunoblot Analysis
  • Support Protocol 4: X‐Gal Overlay Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Cloning of Cub‐PLV and Nub Fusion Constructs

  Materials
  • DNA templates encoding bait and prey proteins of interest
  • mbSUS‐gate vectors of choice (Table 5.27.2)
  • Restriction enzymes ( appendix 1M) appropriate for cutting mbSUS‐gate vector
  • Yeast strains (Table 5.27.1)
    • THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2)
    • THY.AP5 (MATα URA3 leu2 trp1 his3 loxP::ade2)
  • YPAD liquid medium: YPD medium (see recipe) supplemented with 2 mg/liter adenine sulfate
  • LiAc/TE buffer (see recipe)
  • Sheared salmon sperm DNA (unit 4.4)
  • 1× TE buffer (see recipe for 10×)
  • 1 M lithium acetate (LiAc; see recipe)
  • PEG/LiAc mix (see recipe)
  • Synthetic‐complete (SC) medium plates (see recipe):
    • SC/–Leu plates for transformation of THY.AP4 with Cub constructs
    • SC/–Trp plates for transformation of THY.AP5 with Nub constructs
  • Synthetic‐complete (SC) liquid medium (see recipe) containing 200 µg/ml (final concentration) G418:
    • SC/–Leu + 200 µg/ml G418 for THY.AP4 bait CubPLV clones
    • SC/–Trp + 200 µg/ml G418 for clones of THY.AP5 prey
  • Synthetic‐complete (SC) liquid medium (see recipe) without G418
    • SC/–Leu for THY.AP4 bait CubPLV clones
    • SC/–Trp for clones of THY.AP5 prey
  • Zymoprep II Yeast Plasmid Miniprep Kit (Zymo Research)
  • LB medium ( appendix 2A) containing 100 µg/ml ampicillin
  • 28°C shaking incubator
  • Centrifuge
  • 30° and 42°C shaking water baths
  • Additional reagents and equipment for PCR amplification of DNA (CPMB UNIT ), agarose gel electrophoresis ( appendix 1N), isolation of DNA from agarose gels and purification of DNA via affinity columns (CPMB UNIT ), transformation of bacteria by electroporation ( appendix 1E), miniprep isolation of bacterial DNA ( appendix 1J), and DNA sequencing (CPMB Chapter 7)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.

Basic Protocol 2: Characterization of Mating‐Based Split‐Ubiquitin System Bait Fusions

  Materials
  • Yeast strains (Table 5.27.1)
    • THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2)
    • THY.AP5 (MATα URA3 leu2 trp1 his3 loxP::ade2)
  • DNA vectors pNX‐gate32‐3HA (Fig. A) and pNubWT‐Xgate expressing soluble NubG and NubWT peptides, respectively (Table 5.27.2)
  • DNA constructs encoding bait‐Cub‐PLV fusions of interest ( protocol 1)
  • Synthetic complete liquid medium (see recipe):
    • SC/–Leu
    • SC/–Trp
  • Synthetic‐complete (SC) medium plates (see recipe) supplemented with L‐methionine (Met) for repression of bait levels:
    • SC/–Leu plates
    • SC/–Trp plates
    • SC/–Trp, –Leu, –Ura, –Met, supplemented with different concentrations of Met
    • SC/–Trp, –Leu, –Ura, –Ade, –His, –Met, supplemented with different concentrations of Met
    • YPD medium and plates (see recipe)
    • 28°C shaking incubator
    • Centrifuge
  • Additional reagents and equipment for lithium acetate transformation ( protocol 1; also see CPMB UNIT ), replica plating of yeast (CPMB UNIT ), and X‐Gal overlay assay ( protocol 10)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.

Basic Protocol 3: Systematic Split‐Ubiquitin Interaction Tests with Selected Bait and Prey

  Materials
  • Yeast strains (Table 5.27.1)
    • THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2)
    • THY.AP5 (MATα URA3 leu2 trp1 his3 loxP::ade2)
  • DNA constructs encoding bait‐Cub‐PLV fusions ( protocol 1), which passed the quality control in protocol 2
  • NubG‐prey or prey‐NubG fusions of interest ( protocol 1)
  • DNA vectors pNXgate32‐3HA, NubWT‐Xgate and pMetYCgate (Table 5.27.2)
  • Synthetic‐complete (SC) medium plates (see recipe; standard size plates as well as 12 × 12 cm square plates) supplemented with methionine (Met) for repression of bait levels
    • SC/–Leu plates
    • SC/–Trp plates
    • SC/–Trp, –Leu, –Ura, –Met, supplemented with different concentrations of Met
    • SC/–Trp, –Leu, –Ura, –Ade, –His, –Met, supplemented with different concentrations of Met
  • YPD plates (see recipe), square, 12 × 12 cm
  • 28°C incubator
  • Camera or flat‐bed scanner for documenting successful mating on plates
  • Additional reagents and equipment for lithium acetate transformation ( protocol 1; also see CPMB UNIT ), mating and selection of bait and prey fusions ( protocol 2), replica plating of yeast (CPMB UNIT ), and X‐Gal overlay assay ( protocol 10)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.

Alternate Protocol 1: Cloning of Mating‐Based Split‐Ubiquitin Systems Fusions in E. coli

  Materials
  • DNA templates for bait and prey ( protocol 1)
  • pMetYCgate and Nub vectors of choice (see protocol 7 and ) for in vivo cloning of bait and prey B1‐ORF‐B2 PCR products (see protocol 1 for more detail)
  • mbSUS‐gate vectors pMetYCgate, pNXgate32‐3HA and pNubWT‐Xgate as controls (Table 5.27.2)
  • Yeast strains (Table 5.27.1)
    • THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2)
    • THY.AP5 (MATα URA3 leu2 trp1 his3 loxP::ade2)
  • Synthetic‐complete (SC) medium plates (see recipe; standard size plates as well as 12 × 12 cm square plates):
    • SC/–Leu plates
    • SC/–Leu plates + 200 µg/ml G418
    • SC/–Trp plates
    • SC/–Trp plates + 200 µg/ml G418
  • 45% (v/v) glycerol, sterile
  • YPD plates (see recipe), square, 12 × 12 cm
  • Synthetic‐complete (SC) liquid medium:
    • SC/–Leu
    • SC/–Trp
    • SC/–Trp, –Leu, –Ura, –Ade, –His, –Met, supplemented with different concentrations of Met
  • 24‐well plates
  • 96‐well microtiter plates
  • 96‐pin replicator: Multi‐Blot VP407 (V&P Scientific) or Replication System (Nalge Nunc)
  • Spectrophotometer with microtiter plate reader
  • Additional reagents and equipment for cloning of Cub‐PLV and Nub fusion constructs ( protocol 1)
NOTE: All solutions and materials coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

Alternate Protocol 2: Systematic Screening of Sorted Collections of Split‐Ubiquitin Bait and Prey Fusions

  Materials
  • SC/–Trp, –Leu, –Ura microtiter plate arrays with diploid cells ( protocol 3, step 7)
  • SC/–Trp, –Leu, –Ura liquid medium (see recipe)
  • Z buffer (see recipe), ice cold
  • Liquid nitrogen
  • Acid‐washed glass beads (see recipe)
  • Bradford reagent (see recipe)
  • Protein standard: 1 mg/ml BSA in Z buffer
  • ortho‐nitrophenyl‐galactopyranoside (o‐NPG)
  • 1 M Na 2CO 3
  • 28°C shaking incubator
  • Refrigerated centrifuge
  • Spectrophotometer
  • Heating block
  • Additional reagents and equipment for preparing microtiter plate arrays of bait and prey ( protocol 3)

Alternate Protocol 3: Quantitative Analysis of Interactions Between Cub‐PLV and Nub Fusions

  • THY.AP4 and THY.AP5 clones (see protocol 1)
  • Acid‐washed glass beads (see recipe)
  • DNA release solution (see recipe)
  • 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol
  • Isopropanol
  • 80% (v/v) ethanol
  • Additional reagents and equipment for transformation of E. coli ( appendix 1E and CPMB UNIT )

Support Protocol 1: Use of Different Mating‐Based Split‐Ubiquitin System Nub Vectors for Interaction Tests

  Materials
  • THY.AP4 and THY.AP5 clones ( protocol 1) or diploid yeast clones ( protocol 3)
  • Synthetic‐complete (SC) liquid medium (see recipe):
    • SC/–Leu, –Trp, –Ura
    • SC/–Leu
    • SC/–Trp
  • Lysis buffer: 0.2 M NaOH/0.2% (v/v) 2‐mercaptoethanol (prepare fresh)
  • 100% trichloroacetic acid (see recipe)
  • 0.5 M Tris⋅Cl, pH 6.8 ( appendix 2A)
  • Anti‐VP16 (Abcam, http://www.abcam.com) and anti‐HA antibodies (Sigma‐Aldrich)
  • 28°C incubator
  • Refrigerated centrifuge
  • Additional reagents for SDS‐PAGE and immunoblotting (unit 5.19)

Support Protocol 2: Plasmid Rescue from Yeast: “Lazy Bones” Protocol

  • Diploid cells expressing different bait and prey fusions ( protocol 2 and protocol 33)
  • Z buffer (see recipe)
  • 10% (w/v) sodium dodecyl sulfate (SDS) in H 2O
  • 100 mg/ml X‐Gal stock solution in dimethylformamide
  • 50°C water bath
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Dirnberger, D., Unsin, G., Schlenker, S., and Reichel, C. 2006. A small‐molecule–protein interaction system with split‐ubiquitin as sensor. ChemBioChem 7:936‐942.
   Gietz, R.D., Schiestl, R.H., Willems, A.R., and Woods, R.A. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure. Yeast 11:355‐360.
   Griffith, D.A., Delipala, C., Leadsham, J., Jarvis, S.M., and Oesterhelt, D. 2003. A novel yeast expression system for the overproduction of quality‐controlled membrane proteins. FEBS Lett. 553:45‐50.
   Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and Sakaki, Y. 2000. A comprehensive two‐hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. U.S.A. 98:4569‐4574.
   Johnsson, N. and Varshavsky, A. 1994. Split‐ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. U.S.A. 91:10340‐10344.
   Kaiser, C., Michaelis, S., and Mitchell, A. 1994. Methods in Yeast Genetics, a Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York.
   Matsuda, S., Giliberto, L., Matsuda, Y., Davies, P., McGowan, E., Pickford, F., Ghiso, J., Franqione, B., and D'Adamio, L. 2005. The familial dementia BRI2 gene binds the Alzheimer gene amyloid‐beta precursor protein and inhibits amyloid‐beta production. J. Biol. Chem. 280:28912‐28916.
   Obrdlik, P., El‐Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarinoe, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent, V., Blaudez, D., Sanders, D., Revuelta, J.L., Boles, E., Andre, B., and Frommer, W.B. 2004. K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions. Proc. Natl. Acad. Sci. U.S.A. 101:12242‐12247.
   Osborne, A.R., Rapoport, T.A., and van den Berg, B. 2005. Protein translocation by the Sec61/SecY channel. Annu. Rev. Cell Dev. Biol. 21:529‐550.
   Pandey, S. and Assmann, S.M. 2004. The Arabidopsis putative G protein‐coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling. Plant Cell 16:1616‐1632.
   Raquet, X., Eckert, J.H., Müller, S., and Johnsson, N. 2001. Detection of altered protein conformations in living cells. J. Mol. Biol. 305:927‐938.
   Reinders, A., Schulze, W., Kühn, C., Barker, L., Schulz, A., Ward, J.M., and Frommer, W.B. 2002. Intra‐ and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split ubiquitin system and functional assays. Plant Cell 14:1567‐1577.
   Rubio‐Somoza, I., Martinez, M., Abraham, Z., Diaz, I., and Carbonero, P. 2006. Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seeds. Plant J. 47:269‐281.
   Schwacke, R., Schneider, A., van der Graaff, E., Fischer, K., Catoni, E., Desimone, M., Frommer, W.B., Flügge, U.I., and Kunze, R. 2003. ARAMEMNON: A novel database for Arabidopsis integral membrane proteins. Plant Physiol. 131:16‐26.
   Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S. 1998. A genetic system based on split‐ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc. Natl. Acad. Sci. U.S.A. 95:5187‐5192.
   Supply, P., Wach, A., Thinès‐Sempoux, D., and Goffeau, A. 1993. Proliferation of intracellular structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae. J. Biol. Chem. 268:19744‐19752.
   Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshon, D., Narayan, V., Srinivasan, M., Pochrat, P., Quereshi‐Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J.M. 2000. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403:623‐627.
   Ugolini, S. and Bruschi, C.V. 1996. The red/white colony color assays in the yeast Saccharomyces cerevisiae: Epistatic growth advantage of white ade‐8‐18, ade2 cells over red ade2 cells. Curr. Genet. 30:485‐492.
   Wang, B., Pelletier, J., Massaad, M.J., Herscovics, A., and Shore, G.C. 2004. The yeast split‐ubiquitin membrane protein two‐hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum‐associated protein tyrosine phosphatase‐like B. Mol. Cell Biol. 24:2767‐2778.
   Wittke, S., Lewke, N., Müller, S., and Johnsson, N. 1999. Probing the molecular environment of membrane proteins in vivo. Mol. Biol. Cell 10:2519‐2530.
   Zhang, J. and Lautar, S. 1996. A yeast three‐hybrid method to clone ternary protein complex components. Anal. Biochem. 242:68‐72.
Key References
   Johnsson and Varshavsky, 1994. See above.
  Initial description of the split‐ubiquitin.
   Obrdlik et al., 2004. See above.
  Initial description of mbSUS. The paper explains the features of mbSUS and introduces several methods described in this unit.
Internet Resources
  http://www.invitrogen.com
  Commercial resources for Gateway Entry plasmids and for Gateway cloning reagents.
  http://www.biosci.ohio‐state.edu/pcmb/Facilities/abrc/abrchome.htm
  The ABRC database is the source for mbSUS yeast strains as well as the majority of mbSUS vectors.
  http://www.associomics.org/goodies/split_ubiquitin/index.html
  Overview of all split ubiquitin components available at the ABRC database and their ABRC catalog numbers.
  http://www.cbs.dtu.dk/services/TMHMM/
  TMHMM Server v. 2.0. Web server for prediction of transmembrane helices in proteins.
  http://www.enzim.hu/hmmtop/index.html
  HMMTOP: Program for prediction of transmembrane helices and topology of proteins.
  http://www.cbs.dtu.dk/services/SignalP/
  SignalP 3.0 server: Predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms.
  http://www.expasy.ch/tools/#topology
  Tools for topology and signal peptide prediction.
  http://aramemnon.botanik.uni‐koeln.de
  Aramemnon database, providing a comparative view of the topology of plant membrane proteins.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library